Two enzyme systems, brain and ascites tumor hexokinase, and bovine liver fructose-1,6-bisphosphatase will be studied in an attempt to delineate their catalytic mechanisms and their modes of regulation at the molecular level. The kinetic mechanism of fructose-1,6-biphosphatase will be investigated in the nonphysiological direction using dead-end competitive inhibitors of the substrates. Therole of AMP as a regulator of the phosphatase will be studied, and the function of the required divalent metal ion will also be evaluated. Nuclear magnetic resonance studies will be undertaken in an attempt to gain information on the relationships among the substrates at the active site of fructose-1,6-bisphosphatase, and on the proximity of the active and allosteric sites. Studies of the hexokinase enzymes will involve experiments of the reverse reaction in an attempt to evaluate the precise mechanism of inhibition by glucose-6-P and its relief by inorganic orthophosphate. An attempt will be made to investigate the dependence of the kinetic parameters on pH in order to evaluate the functionalgroups associated with catalysis and ligand binding. Experiments involving protein modification and the use of fluorescent probes will be udnertaken with the hexokinases in order to obtain information on the groups associated with catalysis and regulation. Finally, nuclear magnetic resonance studies are contemplated in order to probe the active and regulatory sites of hexokinase.